1. Field of the Invention
This invention relates to an .alpha.-L-arabinofuranosidase effective for hydrolyzing arabinosyl residues from L-arabinose containing polysaccharides and hemicelluloses, and which is stable at high temperatures.
2. Background of the Invention
More than one billion gallons of ethanol are produced annually in the United States, with approximately 95% derived from fermentation of corn starch (Bothast, 1994, Genetically engineered microorganisms for the conversion of multiple substrates to ethanol, Proc. Corn Utilization Conf., National Corn Growers Assoc., St. Louis, Mo.). Various lignocellulosic agricultural residues such as corn fiber, corn stover, straw and bagasse can also serve as low-value and abundant feedstocks for production of fuel alcohol. Currently, the utilization of lignocellulosic biomass to produce fuel ethanol presents significant technical and economic challenges, and its success depends largely on the development of highly efficient and cost-effective biocatalysts for conversion of pretreated biomass to fermentable sugars.
Hemicelluloses, the second most common polysaccharides in nature, represent about 20-35% of lignocellulosic biomass (Wyman, 1994, Bioresourc. Technol., 50:3-16). L-Arabinosyl residues are widely distributed in hemicelluloses as they constitute monomeric and/or oligomeric side chains on the .beta.-(1.fwdarw.4)-linked xylose or galactose backbones in xylans, arabinoxylans and arabinogalactans, and are the core in arabinans forming .alpha.-(1.fwdarw.5)-linkages (Manin et al., 1994, Biochem. J., 302:443-449; Ward and Moo-Young, 1989, CRC Crit. Rev. Biotechnol., 8:237-274). These side chains restrict the enzymatic hydrolysis of hemicelluloses by xylanases (Bachmann and McCarthy, 1991, Appl. Environ. Microbiol., 57:2121-2130). .alpha.-L-Arabinofuranosidases (.alpha.-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55, .alpha.-L-AFase) are exo-type enzymes which hydrolyze terminal non-reducing .alpha.-L-arabinofuranosyl groups from L-arabinose containing polysaccharides. These enzymes can hydrolyze (1.fwdarw.3)- or (1.fwdarw.5) -.alpha.-L-arabinofuranosyl linkages of arabinan or both. The .alpha.-L-AFases are part of microbial xylanolytic systems necessary for complete breakdown of heteroxylans (Bachmann and McCarthy, 1991, ibid; Greve et al., 1984, Appl. Environ. Microbiol., 47:1135-1140; Lee and Forsberg, 1987, Can. J. Microbiol., 33:1011-1016; Poutanen, 1988, J. Biotechnol., 113:15-22).
In recent years, arabinofuranosidases have received much attention because of their practical applications in various agro-industrial processes such as efficient conversion of hemicellulosic biomass to fuels and chemicals, delignification of pulp, efficient utilization of plant materials into animal feed, and hydrolysis of grape monoterpenyl glycosides during wine fermentation (Bezalel et al., 1993, Appl. Environ. Microbiol., 40:57-62; Gilead and Shoham, 1995, Appl. Environ. Microbiol., 61:170-174; Gunata et al., 1990, J. Agric. Food Chem., 38:772-776; Utt et al., 1991, Appl. Environ. Microbiol., 57:1227-1234). There is a need to develop a suitable .alpha.-L-AFase for use in the conversion of hemicellulose to fermentable sugars for the subsequent production of fuel ethanol and other value-added chemicals. .alpha.-L-AFases are produced by several bacteria and fungi but only a few of these enzymes have been purified and characterized (Gilead and Shoham, ibid; Hespell and O'Bryan, 1992, Appl. Environ. Microbiol., 58:1082-1088; Kaji and Tagawa, 1970, Biochim. Biophys. Acta, 207:456-464; Lee and Forsberg, ibid; Tajana et al., 1992, Appl. Environ. Microbiol.; 58:1447-1450). The yeast-like fungus Aureobasidium pullulans has been recognized as an excellent producer of amylases, xylanase and .beta.-glucosidase (Leathers, 1986, Appl. Environ. Microbiol., 52:1026-1030; Saha et al., 1993, Curr. Microbiol., 26:267-273; Saha et al., 1994, Appl. Environ. Microbiol., 60:3774-3780).